Chlamydia trachomatis is the leading cause of infectious blindness and bacterial sexually transmitted infection globally. For the last 20 years, the Gram-negative bacteria comprising the order Chlamydiales were thought to lack peptidoglycan (PG), a bacterial polymer critical for maintaining microbial cell shape and hydrostatic pressure and essential for all bacteria with few exceptions. Despite encoding genes for the complete PG biosynthesis pathway and exhibiting susceptibility to ?-lactam antibiotics, all attempts to detect PG in any chlamydial species have proven unsuccessful, giving rise to the 'chlamydial anomaly'. Research exploring chlamydial PG to date has consisted of complementation studies in E. coli mutants and biochemical analysis of chlamydial proteins expressed in E. coli. While structural models for the assembly of chlamydial PG have been proposed based on these studies, there has been no way to validate their accuracy as there has been no direct evidence that PG exists in this bacterium, until now. By utilizing chemically altered di D-amino acid probes, we have succeeded in labeling the PG of replicating, intracellular Chlamydia trachomatis, allowing us to track cell wall biosynthesis throughout its biphasic life cycle. Probe uptake and incorporation are apparent as early as 8 hours post infection and present throughout development. [Unlike all other bacteria studied to date, Chlamydia does not appear to maintain a PG sacculus (uniform covering over entire microbe) but rather a single PG-ring present at the bacterial division plane, strongly hinting at a role for chlamydial PG in replication. Despite Chlamydia lacking classical PG degradation and recycling pathways, this PG ring appears to be dynamic, with rapid turnover rates even when cell division is artificially arrested with antibiotics.] The long term goal of this project is to fully and completely characterize how Chlamydia creates and re-models its PG and elucidate the consequences of these processes on the mammalian host cell. To meet this goal, the specific aims of this application are 1) to track the biosynthesis and turnover of PG within intracellular and extracellular Chlamydia via use of a novel fluorescent dipeptide labeling strategy, 2) to purify this labeled PG and determine its chemical composition and structure via mass spectrometry, and 3) to examine the eventual degradation and release of chlamydial PG into the host cell cytosol and characterize those enzymes that play a role in this process. Thus, the central hypothesis to be tested in this proposal is that C. trachomatis synthesizes PG and that its ability to synthesize and turnover PG plays an important role in cell division, extracellular survival, and the stimulation of the intracellular innate immune response. The data generated from this research will significantly enhance our understanding of the process of replication in this organism, as well as how the host immune system reacts to the microbe's cellular processes. Additionally, the actual targets of ?-lactam antibiotics on this human pathogen and their effects on PG biosynthesis will finally be elucidated, thereby generating knowledge that has the potential to enhance health and reduce illness for the millions of infected individuals worldwide.